1. Field of the Invention
This invention is directed to an assay for determining the amount of Chinese Hamster Ovary (CHO)-produced tPA present in samples of recombinant human tPA with native sequence or its variants produced in CHO cells.
2. Description of Related Art
Tissue-type plasminogen activators (tPA) are endogenous serine proteases involved in a cascade of events leading to the dissolution of a blood clot (Astrup and Permin, Nature, 159, 681-682 (1947); Camiolo et al., Proc. Soc. Exp. Biol. Med., 138, 277-280 (1971); Collen, J. Cell. Biochem., 33, 77-86 (1987); Hoylaerts et al., J. Biol. Chem., 257, 2912-2919 (1982)). ACTIVASE(copyright) is the recombinant form of human tPA (r-tPA), used in the management of acute myocardial infarction and pulmonary embolism (Grossbard, Pharm. Res., 4, 375-378 (1987)). ACTIVASE(copyright) is also now approved for treating ischemic stroke (Smith et al., Acad. Emergency Medicine, 6(6), 618-25 (1999); Kwiatkowski et al., New Eng. J. Med., 340(23), 1781-1787 (1999)). It is a glycoprotein produced by expressing the complementary DNA (cDNA) for natural human tPA in CHO cells. TNK-tPA is a genetically engineered variant of human tPA cloned and expressed in CHO cells (Keyt et al., Proc. Natl. Acad. Sci USA., 91, 3670-3674 (1994)). Site-directed mutations were introduced at three specific sites of human tPA to create the TNK-tPA variant. They are Thr103 to Asn (T103N), Asn 117 to Gln (N117Q), and Lys-His-Arg-Arg (SEQ ID NO:1) 296-299 to Ala-Ala-Ala-Ala (SEQ ID NO:2) (KHRR296-299AAAA). When compared to tPA, TNK-tPA exhibits similar in vitro biological activity, an increased resistance to plasminogen activator inhibitor and an enhanced fibrin specificity, and is cleared more slowly from plasma (Keyt et al., Proc. Natl. Acad. Sci USA., 91, 3670-3674 (1994); Thomas et al., Stroke, 25:10, 2072-2079 (1994); Benedict et al., Circulation, 92:10, 3032-3040 (1995); Modi et al., Thromb Haemost, 79, 134-139 (1998)). It is currently awaiting regulatory approval as a single bolus administered form of r-tPA. CHO cells biosynthesize endogenous hamster tPA called CHO-PA. CHO-PA has a similar fibrinolytic activity to human tPA as determined by the clot lysis assay. The amino acid sequence of CHO-PA is 80% identical to that of human tPA. Many of the substitutions are semi-conservative such as: Arg less than  greater than Lys, Glu less than  greater than Asp, Phe less than  greater than Tyr, Val less than  greater than Ala, Ile less than  greater than Leu, or Thr less than  greater than Ser. Using a model of the human tPA protease domain based upon the bovine chymotrypsin structure, it is observed that virtually all of the substitutions in CHO-PA are localized at or near the protein surface.
r-tPA, TNK-tPA, and CHO-PA are all single polypeptide chains composed of 527 amino acids with 17 disulfide bonds (Nguyen and Ward, xe2x80x9cStability Characterization and Formulation Development of Altepase, a Recombinant Tissue Plasminogen Activator,xe2x80x9d in Stability and Characterization of Protein and Peptide Drugs: Case Histories, Y. J. Wang, R. Pearlman, eds., (Plenum Press: New York, 1993), pp. 91-135). For all three proteins, the peptide bond between Arg275 and Ile276 is particularly susceptible to protease cleavage. The cleavage results in two fragments: one consisting of the N-terminal 275 amino acids and the other consisting of the C-terminal 252 amino acids. The N-terminal chain contains regions which are homologous to the kringle regions found in plasminogen and prothrombin and, therefore, is often referred to as the xe2x80x9ckringle fragmentxe2x80x9d (Nguyen and Ward, supra; de Vos et al., Biochem., 31, 270-279 (1992)).
The C-terminal chain contains the catalytically active site and, therefore, is commonly referred to as the xe2x80x9cprotease fragmentxe2x80x9d (Pennica et al., Nature, 301, 214-221 (1983)). The cleaved two chains are linked by a single disulfide bond formed between Cys264 and Cys395. The cleaved molecule is commonly referred to as xe2x80x9ctwo-chain tPAxe2x80x9d as opposed to xe2x80x9csingle-chain tPAxe2x80x9d or the intact form.
r-tPA contains four potential sites for N-linked glycosylation identified by the sequence Asn-X-Ser/Thr (Nguyen and Ward, supra). These are Asn117, Asn184, Asn218, and Asn448. r-tPA exists as two glycosylation isozymes designated type I and type II. Type I r-tPA is glycosylated at Asn117, Asn184, and Asn448; whereas type II r-tPA is glycosylated only at Asn117 and Asn448. Asn218 is not glycosylated in either isoform. TNK-tPA has the same glycosylation pattern as r-tPA, except that the Thr103 to Asn and Asn117 to Gln mutations effectively moved the glycosylation site from position 117 to 103 (Keyt et al., supra). The glycosylation pattern for CHO-PA is not fully characterized (Rijken and Collen, J. Biol. Chem., 256: 7035-7041 (1981)).
ACTIVASE(copyright) is a trademark for the recombinant form of human tissue-type plasminogen activator (r-tPA), used in the management of acute myocardial infarction and pulmonary embolism. ACTIVASE(copyright) brand tPA is also now approved for treating ischemic stroke. It is produced by expressing the complementary DNA (cDNA) for natural human tPA in CHO cells (U.S. Pat. No. 5,753,486). TNK-tPA is a genetically engineered variant of r-tPA with enhanced efficacy and lower incidence of bleeding compared with ACTIVASE(copyright) r-tPA. It was created by three site-directed mutations (T103N, N117Q and KHRR (SEQ ID NO:1) 296-299AAAA (SEQ ID NO:2)), and is also cloned and expressed in CHO cells (U.S. Pat. No. 5,612,029). CHO cells biosynthesize endogenous hamster tPA called CHO-PA. The amino acid sequence of CHO-PA is highly homologous (80% identical) to that of r-tPA. All three thrombolytic proteins exist as heterogeneous isoforms, mainly due to proteolysis/hydrolysis and differential glycosylation.
A method for purifying human tPA from CHO-PA is described in U.S. Pat. No. 5,411,864. This method-comprises contacting a fluid containing the human tPA with antibodies specifically binding the corresponding endogenous CHO-PA and recovering the human tPA. Preferably the contacting step involves passing the fluid through a chromatographic bed having the antibodies immobilized thereon.
The development of recombinant DNA-derived protein pharmaceuticals has been facilitated by the introduction of new analytical methods that can be used to characterize protein and/or to demonstrate consistency of manufacture of a protein. Peptide mapping is a key method for monitoring the amino acid sequence and is able to detect small changes in small-to moderate-size proteins, for example, insulin and human growth hormone. The analysis of a much larger protein, e.g., fibrinogen (molecular mass of 350,000), or the heterogeneous glycoproteins, such as antibodies (molecular mass of 150,000), is hindered by the complexity of the range of peptides generated by an enzymatic digestion. Such complexity makes a single reversed-phase high-performance liquid chromatography (RP-HPLC) separation combined with on-line ultraviolet detection of limited utility.
The advent of commercially available combined HPLC and electrospray ionization mass spectromety (LC-ES-MS) systems compatible with convention HPLC has increased the power of peptide mapping considerably (Ling et al., Anal. Chem., 63: 2909-2915 (1991); Guzzetta et al., Anal. Chem., 65: 2953-2962 (1993)). LC-EM-MS in combination with in-source collisionally induced dissociation (CID) has been used effectively to identify sites of N-and O-linked glycosylation (Carr et al., Protein Sci., 2: 183-196 (1993); Huddleston et al., Anal. Chem., 65: 877-884 (1993); Conboy and Henion, J. Am. Soc. Mass Spectrom., 3: 804-814 (1992)).
However, even this technique is limited by insufficient resolution resulting from the large number of very similar peptides caused by variable protein glycosylation and enzymatic digests of moderately sized glycoproteins. It is therefore necessary to employ a range of techniques with orthogonal selectivity to characterize such samples.
The use of combinations of high-performance capillary electrophoresis, HPLC, LC-ES-MS, and matrix-assisted laser desorption ionization-time of flight mass spectrometry has been investigated to allow for characterization of enzymatic digests of underivatized glycoprotein samples, as exemplified by DSPAxcex11, a single-chain plasminogen activator derived from vampire bat salivary glands (Apffel et al., J. Chromatography A, 717: 41-60 (1995)). It was concluded that these four techniques are highly complimentary techniques for examining glycoproteins. Nonetheless, the authors acknowledge that more work needs to be done to improve the power of this approach, and that high-yield concentration steps will be required due to extensive carbohydrate heterogeneity.
There is a need for a technique to monitor the relative and absolute amounts of CHO-PA present after a purification procedure for tPA is carried out, such as the one reported in U.S. Pat. No. 5,411,864, supra.
Accordingly, a reversed-phase HPLC method was developed herein for the analysis of the three thrombolytic molecules, CHO-tPA, recombinant human tPA with native sequence, and TNK-tPA. This method not only has the ability to resolve human tPA and/or TNK-tPA from CHO-PA, but also is capable of identifying and quantifying different isoforms of each molecule.
Specifically, the present invention provides a process for monitoring the effectiveness of a purification process in removing plasminogen activator (PA) endogenous to Chinese hamster ovary (CHO) cells from a sample containing human tPA or variants thereof, which process comprises incubating the sample with a protease capable of specifically cleaving the Arg275-Ile276 bond of human wild-type tPA and then with denaturing and reducing agents in amounts effective to reduce the disulfide bonds of human wild-type tPA; subjecting the sample to a reversed-phase high-performance liquid chromatography step, and analyzing the elution profile from the chromatography step for the amount of PA endogenous to the CHO cells present therein.